Cell cycle checkpoints ensure genomic integrity during cell proliferation (1). Loss or alteration of these cell cycle check-points can lead to changes in cell proliferation that eventually result in disease states. A leading disease in skin tissue is cancer due to UV-radiation exposure (2). Fortunately, eukaryotic cells possess a mechanism known as the "damage response", a transcriptional response triggered by DNA damage. This induction includes the expression of various genes involved in DNA repair, DNA synthesis and cell cycle checkpoint. Two gene products shown to play a protective role in the cellular response to DNA damage are p53 and its downstream transcriptional 'target', p21WAF1/CIP1 (3). We hypothesize that p21WAF1/CIp1 plays an important role in normal human keratinocyte cell cycle arrest following DNA damage by regulating cell cycle components involved in G1/S and G2/M checkpoints. Also, given the association between G1 arrest and cell differentiation, we hypothesize the p21WAF1/CIP1 may be recruited to link differentiation signals to G1 arrest in human keratinocytes (NHEKs). To test these hypotheses the following Specific Aims are proposed: 1. To determine the role of p21WAF1/CIP1 in DNA-damage induced growth arrest and apoptosis in NHEKs. a) Cell cycle analysis will be performed to determine the modulatory effect of DNA damage on cell cycle transition in NHEKs. b) Complex formation between p21WAF1/CIP1 and specific cell cycle components will be assessed at determined times following DNA damage. In parallel, direct assays to measure cyclin-dependent kinase activity will be performed. 2. To determine the role of p21WAF1/CIP1 in human keratinocyte differentiation. a) Cell cycle analysis will be performed following Ca2+-induced differentiation of NHEKs to determine time points to assay p21WAF1/CIP1 activity. Appropriate markers of keratinocyte differentiation will be examined in parallel. b) Complex formation between p21WAF1/CIP1 and specific cell cycle components will be assessed at various times following induction of differentiation. In parallel, direct assays to measure cyclin-dependent kinase activity will be performed. Further characterization of DNA damage and differentiation pathways in normal skin keratinocytes will lend insight to the design of novel therapeutic strategies for chemoprevention and chemotherapy of skin cancer and hyperproliferative skin diseases.